The use of ionic liquids as crystallization additives allowed to overcome nanodrop scaling up problems: A success case for producing diffraction-quality crystals of a nitrate reductase

Abstract

The native structure of the heterodimeric periplasmic nitrate reductase (NapAB) from Cupriavidus (C.) necator was solved at 1.5 Å resolution, using one single crystal obtained at the robot facility at the EMBL, Grenoble. The reaction mechanism for this family of proteins was recently revised, based on new crystallographic evidence, and new structural studies are required to clarify this new mechanistic implication. Several nanodrop crystallization trials yielded microcrystals of the C. necator NapAB. However, scale-up attempts systematically failed and did not yield any suitable crystals. Only with the use of ionic liquids (IL) were we able to grow, in a reproducible manner, larger crystals, which diffracted X-rays to 1.7 Å resolution. By using the IL [C₄mim]Cl as a crystallization additive, we achieved reproducibility in obtaining good quality crystals. Although no IL molecules could be identified in the electron density maps, the crystals grown in the presence and absence of IL have large differences in cell constants. This is the first report of the use of IL for a difficult crystallization problem. The procedure now reported can be applied for crystal optimization such as size increase or improvement of fine needles, as well as for scaling-up crystallization conditions from nanolitre to microlitre drop volumes.

Introduction

The periplasmic nitrate reductase (NapAB) from Cupriavidus (C.) necator is a molybdopterin-dependent enzyme, belonging to the DMSO reductase family of molybdenum enzymes. These enzymes take part in the bacterial denitrification pathway, catalyzing the two-electron reduction of nitrate to nitrite, with release of one water molecule. C. necator NapAB is heterodimeric, containing a catalytic subunit (91 kDa), which harbours the molybdenum active site and a [4Fe–4S] cluster, and a 17 kDa subunit, which contains two hemes involved in electron transfer. The crystal structures of three different periplasmic nitrate reductases have been determined [1], [2], [3], and the corresponding 3D structures have allowed to correlate the molecular structures with the enzymatic function as well as with detailed spectroscopic studies (EPR and EXAFS) of the catalytic molybdenum active site [4]. The generally accepted reaction mechanism for the reduction of nitrate was deduced based on the first crystal structure reported for the monomeric NapA from Desulfovibrio (D.) desulfuricans ATCC 27774 [1]. Recent detailed crystallographic studies of the same enzyme have shown that the coordination sphere is different from what was believed [5]. These results have revealed the presence of an unexpected sulphur ligand coordinating the Mo atom instead of oxygen. Furthermore, this crystal structure revealed that the sulphur atom establishes a partial disulphide bond with the Mo-coordinating cysteine. These results have obvious implications for the enzymatic mechanism that has to be reinvestigated. Possible alternative reaction mechanisms were proposed based on the crystallographic results as well as on the chemistry of molybdenum compounds [5]. However, further experimental data are necessary to corroborate the mechanistic proposals. Experiments should include crystallographic studies of different forms of the enzyme, namely of reduced forms as well as of complexes with substrate analogues and inhibitors. These structural results, supported by additional biochemical evidence and spectroscopic data, should contribute to the understanding and definition of a structure-supported reaction mechanism for nitrate reductases. Further studies of C. necator NapAB have however been hampered due to the lack of suitable crystals, in part caused by low expression levels and difficult purification. Owing to the initial limited amounts of protein, we took advantage of the crystallization robot facility at the high throughput crystallization laboratory (EMBL, Grenoble). Some small but well diffracting crystals were obtained and used to solve the structure of the oxidised form of the enzyme [6]. Since more crystals were needed for mechanistic studies, we attempted to scale up from 200 nl drops to 1 μl. However, these trials were unsuccessful.

In the search for new efficient protocols for crystallizing C. necator NapAB, water-soluble ionic liquids (IL) were tested as additives to the crystallization conditions. These relatively novel compounds are organic salts, liquid at room temperature and are typically composed of an organic cation (e.g. 1-butyl-3-methylimidazolium [C₄mim]) and any of a variety of counter-anions (e.g. Cl⁻, PF₆⁻, BF₄⁻, 2(2-methoxyethoxy) ethyl sulphate [MDEGSO₄]). Their properties, such as solubility, polarity and hydrophilicity, may be tuned by selecting the appropriate anion or cation, rendering them remarkably versatile for many applications. IL interact with proteins, replacing for some of the interactions with water, therefore they may act as additives (or precipitants) for macromolecular crystallization [7]. N′-alkyl-N-methylimidazolium chlorides (as [C₄mim]Cl⁻) have been studied as refolding additives [8], and have been classified as preferentially bound and slightly to moderately chaotropic agents. The effect of these IL was correlated with the hydrophobicity of the substituted imidazolium cations. For example ionic liquids with longer N′-alkyl chains (e.g. hexyl, [C₆mim]Cl) can have properties similar to cationic detergents and can be considered as additives in membrane protein crystallization.

Despite the large number of studies involving ionic liquids in protein stabilization [8], [9], only two reports of their use in protein crystallization were published to date [10], [11]. The first report of the use of ethyl ammonium nitrate in the crystallization of lysozyme was in 1999 [10]. The second, dated 2007, describes the use of IL for crystal improvement of four proteins (canavalin, β-lactoglobulin B, xylanase and glucose isomerase) [11]. However, this study is performed on model proteins, which posed no crystallization problems. Furthermore, only crystal morphology was assessed, and no details with respect to the diffraction quality of the improved crystals were given. Very recently, a third publication appeared [12] in which the authors performed similar experiments as Pusey et al. [11] but also for model proteins (lysozyme, catalase, myoglobin, trypsin, xylanase and glucose isomerase) and improvement in crystal size was only found for lysozyme and trypsin.

The results now reported are for a real case scenario of a difficult crystallization problem. The nanodrop conditions could only be reproduced in larger volume drops only when IL were used as additives. We could thus obtain diffraction quality crystals in a reproducible manner. The consequences of these results are twofold: (a) reproducibility of good quality crystals, which will allow pursuing the mechanistic studies of periplasmic nitrate reductases and (b) development of a strategy that can be applied to many other cases, such as the size increase of microcrystals, and crystal diffraction improvement or for the scale-up of crystallization conditions from nanolitre to microlitre drop volumes.